Enhanced detection system and method

ABSTRACT

An enhanced detection system can eliminate use of a sheath fluid by selecting which particles that pass through an sensing region to detect parametric characteristics thereof based upon position of each particle while it is in a sensing region relative to one or more predetermined positions, such as an in-focus position relative to one or more light beams directed into the sensing region, to enhance accuracy and robustness of particle parametric characteristics detection.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority benefit of provisional application Ser.No. 60/888,706 filed Feb. 7, 2007, the content of which is incorporatedin its entirety.

FIELD OF THE INVENTION

Generally, the present invention relates to flow cytometry.

BACKGROUND OF THE INVENTION

With conventional flow cytometers, particles, such as cells, are alignedand carried along ideally in a single file arrangement within a streamof clear fluid, also known as a sheath fluid, to pass before one or morebeams of light in an sensing region for subsequent detection of variousparametric characteristics to classify, categorize, quantify orotherwise detect one or more aspects of the particles. This sheath fluidguides the particles substantially along a desired trajectory to keepthe particles in-focus in the sensing region relative to one or morebeams of light for subsequent sensing by detectors and automatedquantification of cells according to predetermined parametriccharacteristics. Without the sheath fluid, particles while in thesensing region may not be in a proper in-focus position relative to thebeams of light directed into the sensing region so that detection datawould be collected regarding out-of-focus and in-focus particles. Thedetection data collected regarding the out-of-focus particles would lackaccuracy and consequently harm the overall integrity of the datacollected.

Due to the single-file nature of the particles passing in the sheathfluid, for each particle passing through or in the vicinity of one ormore of the light beams in an sensing region, there is generally littleor no surrounding particles so that there is little background lightscatter or fluorescence to interfere with detection of the predeterminedone or more parametric characteristics associated with the particle sothat each of the particles can be considered as in-focus while in thesensing region with respect to the one or more beams of light involved.Unfortunately, the need for both clean sheath fluid and a highly stablestream greatly complicates the fluidics of these systems. As aconsequence, setup and maintenance of these systems while measurementsare being performed is very labor intensive. In addition, related systemdesign such as involving sheath fluid management and sample injectionconstitute a significant proportion of the complexity found withconventional flow cytometry systems.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a schematic diagram of an enhanced detection system accordingto the present invention.

FIG. 2 is a perspective view of a portion of the detection system ofFIG. 1.

FIG. 3A is a side elevational schematic view of an in-focus case of animplementation of the detection system of FIG. 1 in operation.

FIG. 3B is a side elevational schematic view of an out-of-focus case ofan implementation of the detection system of FIG. 1 in operation.

FIG. 4 is a first collection of plots showing detection results of animplementation of the enhanced detection system of FIG. 1.

FIG. 5 is a second collection of plots showing detection results of animplementation of the enhanced detection system of FIG. 1.

DETAILED DESCRIPTION OF THE INVENTION

An enhanced particle parametric characteristics detection system andmethod has various implementations that reduce or eliminate conventionalreliance on a sheath fluid. The enhanced detection system selects whichparticles that pass through an sensing region to detect parametriccharacteristics thereof based upon position of each particle while it isin the sensing region relative to one or more predetermined positions,such as an in-focus position relative to one or more light beamsdirected into the sensing region, to enhance accuracy and robustness ofparticle parametric characteristics detection. Through use of positiondetection while each particle is in the sensing region, the enhanceddetection system can sense when a particular particle is in-focus orout-of-focus relative to the light beams directed into the sensingregion used to detect predetermined characteristics of the particles.Detection data regarding in-focus particles and out-of-focus particlescan be then discriminated. For instance, detection data regardingin-focus particles can then be saved and the detection data regardingout-of-focus particles can be disregarded. It can be desirable todisregard detection data for out-of-focus particles since particlepositioning does not allow for accurate collection of data regardingthese out-of-focus particles.

In other words, particle position information can be used to modify ordisregard detection of one or more predetermined parametriccharacteristics associated with particles that do not follow a desiredtrajectory or one of a collection of trajectories while in an sensingregion. In some implementations, particle position signals generatedthrough electronic circuitry are inputted to software-based filtering todisregard detection of one or more predetermined parametriccharacteristics for particles that do not follow a predeterminedtrajectory and/or one of a collection of trajectories while in thesensing region. As a consequence, in these implementations, detectorsare used to detect results caused by one or more beams of light beingdirected onto only those particles of a flow of particles that are in adesignated position while in the sensing region, such as a position ofin-focus relative to those one or more beams of light.

In some implementations, the presence of a properly positioned particle,such as an in-focus particle, having a predetermined trajectory while inan observation can be ascertained with a sensing system based uponconfocal microscopy. Other implementations can use other ways todetermine which particles in a flow of particles occupy a predeterminedposition, such as an in-focus position, while in a sensing region.Confocal microscopy teaches that images related to properly positionedparticles, such as in-focus particles, will be focused to pass throughan aperture, such as a pinhole mirror, which can then be received by asingle selectively positioned detector, while images related toimproperly positioned particles, such as out-of-focus particles, will beunfocused thereby casting light in a plurality of directions that canthen be detected by appropriately placed detectors. Signals sent fromthe detectors can then be analyzed to ascertain status regarding whethereach particle in a flow of particles is positioned while in a sensingregion either in an in-focus position or an out-of-focus position.

Implementations of the enhanced detection system can be operated withoutneed of a sheath fluid and thus can be used to directly analyzeenvironmental samples such as ocean, lake, and stream water. Theenhanced detection system provides an added convenience withcompactness, few moving parts, relatively low power requirements, andease of operation that can be exploited in many applications such asfound with use in remote locations and unattended operation. Anotheraspect of the enhanced detection system is that the technique may beextended to incorporate other cytometric measurements involvingadditional lasers, scattering, apertures, fluorescence bands, and/orpolarization measurements.

An exemplary enhanced detection system 100 is depicted in FIG. 1 ashaving a first laser 102 of a first wavelength (e.g. 532 nm) and asecond laser 104 of a second wavelength (e.g. 638 nm). The enhanceddetection system 100 includes a dichroic mirror 106 (selectively passeslight of the first wavelength and reflects light of the secondwavelength), a prism (or mirror or other reflector) 108 and a microscopeobjective 110 (uses confocal illumination) to produce two (nearly)co-linear laser lines to illuminate a sensing region 111 shown ascontaining an in-focus particle 112.

The enhanced detection system 100 includes a lens 114 to focus lightfrom the objective 110 that has impinged upon material in the sensingregion 111, such as the in-focus particle 112. The focus light isdirected to an aperture 116 (with a proximate side 116 a and a distalside 116 b) to image the sensing region 111 under a limited field ofview. The aperture 116 is coupled with a structure 117 having aproximate side 117 a and a distal side 117 b. Located adjacent toportions of the distal side 116 b of the aperture 116 and the distalside 117 b of the structure 117 are a first peripheral prism 118 a (orother such diverter) and a second peripheral prism 118 b.

The enhanced detection system 100 includes a first peripheral detector120 a (e.g. photomultiplier tube), and a second peripheral detector 120b located to receive light transmitted through the first peripheralprism 118 a and the second peripheral prism 118 b, respectively. Theenhanced detection system 100 further includes a first parametriccharacteristics detector 122 and a second parametric characteristicsdetector 124 each receiving light of different wavelengths according toa second dichroic mirror 126. In aggregate, these components providecytometric information about cells passing through the sensing region bymeasuring the scattered light and fluorescence at two (or more)different excitation wavelengths. By adding more apertures, thefunctionality of the sensor can be extended by adding a number of laserlines to excite other fluorophores.

When a particle in the sensing region 111 is in-focus, light from thefirst laser 102 and the second laser 104 will be directed straightthrough the aperture 116 so that substantially only the first parametriccharacteristics detector 122 and the second parametric characteristicsdetector 124 will sense the light as shown in FIG. 1.

The enhanced detection system 100 is also shown to include a CCD camera128 that allows visualization of both the sensing region 111 and laserillumination for such things as alignment. Additionally, the CCD camera128 gives the option of imaging larger particles.

The aperture 116, the structure 117, the first peripheral prism 118 a,and the second peripheral prism 118 b are shown in FIG. 2 in an enlargedview. The depicted implementation includes the aperture 116 as elongatedwith portions of the first peripheral prism 118 a and the secondperipheral prism 118 b located adjacent to end portions of the aperture.Light from a properly focused particle passes through the aperture 116and on to the first parametric characteristics detector 122 and thesecond parametric characteristics detector 124.

Light from particles that are out-of-focus and therefore out of thefocal area is cast onto the first peripheral prism 118 a and the secondperipheral prism 118 b that overhang each end edge of the elongateddimension of the aperture 116 to be directed to the first peripheraldetector 120 a and the second peripheral detector 120 b, respectively.Signals generated by the first peripheral detector 120 a and the secondperipheral detector 120 b can be used by the controller 130 gate outmeasurements made by the first parametric characteristics detector 122and the second parametric characteristics detector 124 related toparticles that were out-of-focus while in the sensing region 111.

As discussed above, as shown in FIG. 3A, when light is directed straightthrough the aperture 116, it is received by the first parametriccharacteristics detector 122 (and the second parametric characteristicsdetector 124 if the second chronic mirror 126 is used). If light isdirected through the aperture 116 on an angle due to a particle beinglocated in an out-of-focus position in the sensing region 111, as shownin FIG. 3B, the light will be further directed to the first peripheraldetector 120 a through the first peripheral prism 118 a and/or thesecond peripheral detector 120 b through the second peripheral prism 120b.

Measurement results are shown in FIG. 4 for an implementation of theenhanced detection system 100 using a 488 nm or a 357 nm laser as theexcitation source and the prism 108 to steer laser light through a 20×or 10× version of the objective 110. Scattered and fluorescent lightemission from particles in a typical flow cytometer stream was collectedusing the enhanced detection system 100. Depicted measurements are for1.0 □m polystyrene beads both in and out of the focal area. Out-of-focusparticle measurements were simulated by moving the stream from side toside, as well as forwards and back. As illustrated in FIG. 4 clockwisefrom top left: no movement, side to side 0.001″ each way, forward0.001″, and back 0.001″. Fluorescent yellow-green 1.0 □m beads, 10×objective, 488 nm 200 mW excitation.

Ability of the enhanced detector 100 to measure real cell populations isdepicted in FIG. 5. A mix of marine algae was injected into a typicalflow cytometer stream with the sample pressure boosted to a level wherethe sample flow was unsteady and not necessarily in the focal area of a20× version of the objective 110. Light signals detected by the enhanceddetection system 100 (the first peripheral detector 120 a being labeledas D1 and the second peripheral detector 120 b being labeled as D2) thatare common to both peripheral detectors are in the side-side center ofthe sheath fluid. The proximal and distal position of a particle fromthe in-focus position can be determined from the relative levels oflight that passes directly through the aperture 116 as sensed by thefirst parametric characteristics detector 122 and the second parametriccharacteristics detector 124 and the light that is cast onto the firstperipheral prism 118 a as sensed by the first peripheral detector 120 aand the second peripheral prism 118 b as sensed by the second peripheraldetector 120 b in this method for determining position of particle withrespect to optimal focal area. Epi-illuminescent measurement (BSC) ofSynechococcus spp., Thalassiosira pseudonana, and Thalassiosiraweisfloglii marine algae are shown; 300 mW 457 nm excitation, 20×objective. The gated region at the left is used to filter out eventsfrom particles that are not properly positioned for a measurement. Gateddata showing chlorophyll versus phycoerythrin are shown at bottom right.

It will be readily understood by those persons skilled in the art thatthe present invention is susceptible of broad utility and application.Many embodiments and adaptations of the present invention other thanthose herein described, as well as many variations, modifications andequivalent arrangements, will be apparent from or reasonably suggestedby the present invention and the foregoing description thereof, withoutdeparting from the substance or scope of the present invention.Accordingly, while the present invention has been described herein indetail in relation to its preferred embodiments, it is to be understoodthat this disclosure is only illustrative and exemplary of the presentinvention and is made merely for purposes of providing a full andenabling disclosure of the invention. The foregoing disclosure is notintended or to be construed to limit the present invention or otherwiseto exclude any such other embodiments, adaptations, variations,modifications and equivalent arrangements, the present invention beinglimited only by the claims filed and the equivalents thereof.

1. A system comprising: a laser configured to output light having afirst wavelength; an objective configured to direct light onto a sensingregion, the sensing region including a first position and a secondposition; an aperture having a proximate side and a distal side, theproximate side being closer to the objective than the distal side, theaperture positioned to receive light from the objective; a diverterpositioned to substantially redirect a first light having a firstdirection to a second direction, the first light having been receivedthrough the aperture from the proximate side having the first direction,the diverter positioned to allow a second light having been receivedthrough the aperture from the proximate side having a third direction toremain in the third direction; a first detector positioned to receive atleast a portion of the first light having the second direction; and asecond detector positioned to receive at least a portion of the secondlight having the third direction.
 2. The system of claim 1 furtherincluding a controller configured to receive a first signal from thefirst detector when the at least a portion of the first light is beingreceived by the first detector and to receive a second signal from thesecond detector when the at least a portion of the second light is beingreceived by the second detector
 3. The system of claim 2 wherein thecontroller is configured to record data associated with the secondsignal received from the second detector during an absence of the firstsignal being received by the first detector.
 4. The system of claim 2wherein the controller is configured to refrain from recording dataassociated with the second signal received from the second detectorwhile the first signal is being received from the first detector.
 5. Thesystem of claim 1 wherein the diverter is a prism.
 6. The system ofclaim 1 wherein the first detector and the second detector are photomultipliers.
 7. The system of claim 1 wherein the first position of thesensing region is associated with a particle being out-of-focus with thefirst light having the first direction.
 8. The system of claim 1 whereinthe second position of the sensing region is associated with a particlebeing in-focus with the second light having the third direction.
 9. Thesystem of claim 1 further including a reflector to redirect at least aportion of the light from the laser along a path toward the objective.10. The system of claim 1 further including a second laser configured tooutput light having a second wavelength.
 11. The system of claim 10further including a dichroic mirror positioned and configured to passlight of the first wavelength and reflect light of the second wavelengthalong a path leading toward the objective.
 12. The system of claim 10further including a third detector and a dichroic mirror positioned andconfigured to pass a portion of the second light having the firstwavelength in the third direction toward the second detector and reflecta portion of the second light having the second wavelength in a fourthdirection toward the third detector.
 13. The system of claim 12 whereinthe controller is configured to receive signals from the third detectorand to record data associated with signals from the third detectorduring an absence of the first signal being received from the firstdetector.
 14. The system of claim 13 wherein the controller isconfigured to refrain from recording data associated with signalsreceived from the third detector while the first signal is beingreceived by the first detector.
 15. The system of claim 12 wherein thefirst wavelength is 532 nm and the second wavelength is 638 nm.
 16. Thesystem of claim 1 wherein the objective uses confocal illumination. 17.The system of claim 1 further including a second diverter, the seconddiverter positioned to substantially redirect a portion of the firstlight having the first direction to a fifth direction, the seconddiverter positioned to allow the second light having been receivedthrough the aperture from the proximate side having a third direction toremain in the third direction;
 18. The system of claim 17 furtherincluding a fourth detector positioned to receive a portion of the firstlight having the fifth direction.
 19. A system comprising: an aperturehaving a proximate side and a distal side, the proximate side beingconfigured to be positioned closer to an objective than the distal side;a first prism, a portion thereof located adjacent a portion of theaperture to redirect light received through the aperture from a firstdirection to a second direction and to remain unengaged with lightpassing through the aperture in a third direction; a first detectorpositioned to receive light in the second direction; a second detectorposition to receive light in the third direction; and a controllerconfigured to receive signals from the first detector and the seconddetector, the controller configured to record data associated withsignals received from the second detector with an absence of signalsbeing received from the first detector and to refrain from recordingdata associated with signals received from the second detector whensignals are being received from the first detector.
 20. The system ofclaim 19 further including a second prism, a portion thereof locatedadjacent a second portion of the aperture to redirect the light receivedthrough the aperture from the first direction to a fourth direction andto remain unengaged with light passing through the aperture in the thirddirection and including a third detector positioned to receive light inthe fourth direction wherein the controller is configured to receivesignals from the third detector and to record data associated withsignals from the second detector with an absence of signals beingreceived from the third detector and to refrain from recording dataassociated with signals received from the second detector when signalsare being received from the third detector.
 21. A method comprising:outputting light having a first wavelength; directing light onto asensing region, the sensing region including a first position and asecond position; receiving light fro the sensing region through anaperture having a proximate side and a distal side, the proximate sidebeing closer to sensing region than the distal side; redirecting a firstlight having a first direction to a second direction, the first lighthaving been received through the aperture from the proximate side havingthe first direction; allowing a second light having been receivedthrough the aperture from the proximate side having a third direction toremain in the third direction; detecting at least a portion of the firstlight having the second direction; and detecting at least a portion ofthe second light having the third direction.
 22. The method of claim 21further including receiving a first signal upon detecting the firstlight and receiving a second signal upon detecting the second light 23.The method of claim 22 including recording data associated with thereceiving of the second signal during an absence of the receiving thefirst signal.
 24. The method of claim 22 including refraining fromrecording data associated with the receiving the second signal while thereceiving the first signal.
 25. The method of claim 21 wherein theredirecting is through a prism.
 26. The method of claim 21 wherein thedetecting the first light and the second light is by photo multipliers.27. The method of claim 21 wherein the first light is from a reflectionfrom a particle in an out-of-focus position.
 28. The method of claim 21wherein the second light is from a reflection from a particle in anin-focus position.
 29. The method of claim 21 further includingredirecting at least a portion of the light from a laser along a pathtoward an objective.
 30. The method of claim 21 further includingoutputting light having a second wavelength.
 31. The method of claim 30further including passing light of the first wavelength and reflectinglight of the second wavelength along a path leading toward theobjective.
 32. The method of claim 30 further including passing aportion of the second light having the first wavelength in the thirddirection and reflecting a portion of the second light having the secondwavelength in a fourth direction.